Project Summary A complete understanding of the genetic machinery underlying tissue regeneration is desirable for further advances in regenerative and diagnostic medicine. While some parts of the gene regulatory networks underlying organ and tissue regeneration are understood, we lack basic knowledge regarding if/how such data are applicable to other organ and tissue types, and the levels of involvement from the different participating cell layers. The overall goal of the proposed work is to dissect tooth regeneration in zebrafish and stickleback fish, which are well suited to such inquiries. While most mammalian teeth are replaced only one or zero times during their lives, the majority of other vertebrate species (fishes, amphibians, reptiles, etc) regenerate their teeth constantly. Despite this fact, the genetic cascades underlying dental stem cells or stem-cell like progenitors remain largely mysterious. Fish offer powerful genetic advantages including large numbers of rapidly developing offspring, external fertilization, and highly efficient transgenesis and genome editing methods. Using an unbiased approach in sticklebacks, which constantly replace their teeth, the Miller lab identified Bone morphogenetic protein 6 (Bmp6) attenuation as a genetic event correlated with tooth regeneration. This mechanism mimics mammalian hair regeneration, wherein BMP attenuation is crucial for a shift from mature hair to replacement hair initiation. Furthermore, RNA-seq has identified strong positive and negative correlations between stickleback Bmp6 mRNA levels and the expression of genes likely involved in tooth regeneration, many of which are also implicated in hair regeneration, such as Lgr6 and Wnt pathway genes. Thus, as in hair, Bmp attenuation appears to be a key event in the initiation of replacement teeth. These data suggest that these regeneration programs use a shared gene regulatory network that predates our last aquatic relatives and their continuously replaced teeth. Using this wealth of preliminary data, I will look both upstream and downstream of Bmps in order to further elucidate the genetic pathways responsible for tooth regeneration and to test the hypothesis that teeth and hair regenerate from a shared genetic program. To this end, I will identify gene expression patterns during tooth regeneration associated with putative stem cell niches (Specific Aim 1), test if conserved genetic mechanisms regulate and transduce Bmp6 signals in teeth, and whether these are associated with niche maintenance (Specific Aim 2), and assay the role of Bmp signaling activity in the activation of Wnt signaling in the niche (Specific Aim 3). Comparing these data to other organ systems, namely hair, will fill critical gaps in our knowledge regarding tissue regeneration, paving the way for future attempts to regenerate human teeth and other tissues in vitro and ultimately in vivo.